The impact of the polymerase chain reaction (PCR) on biological research is perhaps most clearly evidenced by the number and variety of specific applications of this technique. The power and versatility of PCR amplification has transformed this method into a standard tool in molecular biology research and created an ever increasing demand for PCR instrumentation and reagents. An unfortunate corollary of PCR is that errors resulting from properties inherent to the reagents are amplified and unwittingly interpreted as the target sequence.
One prevalent source of error is DNA polymerase catalyzed non-templated addition of a nucleotide to the 3' hydroxyl terminus of duplex PCR products. This activity has been reported for DNA polymerases from Thermus aquaticus, polymerase .alpha. from chick embryo, rat polymerase .beta., reverse transcriptase from avian myeloblastosis virus, and DNA polymerase I from Saccharomyces cerevisiae. Clark, Nucleic Acids Research, 16(20):9677-9686 (1988). In the presence of all four deoxynucleotidetriphosphates (dNTPs), these DNA polymerases differ in the efficiency with which particular dNTPs are added but generally display a preference for non-templated addition of dATP. Clark reported that template independent addition required a duplex DNA substrate but stated that adenylation cannot involve the use of coding information from the template strand. More recently, it was reported that Taq polymerase is relatively resistant to adding an extra nucleotide on a 3' terminal adenosine, and relatively efficient at adding an adenosine residue on a 3' terminal cytosine. Hu, DNA and Cell Biology, 12(8):763-770 (1993).
At best, non-templated 3' adenylation of an otherwise blunt-ended duplex may result in inefficient cloning. More disturbing, however, is its impact on genotyping, where artifactual variations in marker size may adversely impact interpretations of family relationships, medical diagnosis, and forensics. Moreover, full automation of genotyping has been hampered by the necessity of manually editing collected data to correct for allele misidentification due to the unpredictability of non-templated nucleotide addition.
What is needed in the art is a means to control (i.e., reduce or more consistently promote) non-templated nucleotide addition. Further, a method to facilitate template independent addition is needed to exploit the advantageous properties of 3' adenylated PCR products. Quite surprisingly, the subject invention provides these and other advantages.